Urine-derived stem cells genetically modified with IGF1 improve muscle regeneration.

OBJECTIVE: In this study we aimed to determine the impact of human urine derived stem cells (USC) and genetically modified USC that were designed to overexpress myogenic growth factor IGF1 (USCIGF), on the regenerative capacity of cardiotoxin (CTX)-injured murine skeletal muscle. METHODS: We overexpressed IGF1 in USC and investigated the alterations in myogenic capacity and regenerative function in cardiotoxin-injured muscle tissues. RESULTS: Compared with USC alone, USCIGF1 activated the IGF1-Akt-mTOR signaling pathway, significantly improved myogenic differentiation capacity in vitro, and enhanced the secretion of myogenic growth factors and cytokines. In addition, IGF1 overexpression increased the ability of USC to fuse with skeletal myocytes to form myotubes, regulated the pro-regenerative immune response and inflammatory cytokines, and increased myogenesis in an in vivo model of skeletal muscle injury. CONCLUSION: Overall, USC genetically modified to overexpress IGF1 significantly enhanced skeletal muscle regeneration by regulating myogenic differentiation, paracrine effects, and cell fusion, as well as by modulating immune responses in injured skeletal muscles in vivo. This study provides a novel perspective for evaluating the myogenic function of USC as a nonmyogenic cell source in skeletal myogenesis. The combination of USC and IGF1 expression has the potential to provide a novel efficient therapy for skeletal muscle injury and associated muscular defects in patients with urinary incontinence.

Stem cell therapy combined with controlled release of growth factors for the treatment of sphincter dysfunction.

Sphincter dysfunction often occurs at the end of tubule organs such as the urethra, anus, or gastroesophageal sphincters. It is the primary consequence of neuromuscular impairment caused by trauma, inflammation, and aging. Despite intensive efforts to recover sphincter function, pharmacological treatments have not achieved significant improvement. Cell- or growth factor-based therapy is a promising approach for neuromuscular regeneration and the recovery of sphincter function. However, a decrease in cell retention and viability, or the short half-life and rapid degradation of growth factors after implantation, remain obstacles to the translation of these therapies to the clinic. Natural biomaterials provide unique tools for controlled growth factor delivery, which leads to better outcomes for sphincter function recovery in vivo when stem cells and growth factors are co-administrated, in comparison to the delivery of single therapies. In this review, we discuss the role of stem cells combined with the controlled release of growth factors, the methods used for delivery, their potential therapeutic role in neuromuscular repair, and the outcomes of preclinical studies using combination therapy, with the hope of providing new therapeutic strategies to treat incontinence or sphincter dysfunction of the urethra, anus, or gastroesophageal tissues, respectively.

Freezing Biological Time: A Modern Perspective on Organ Preservation.

Transporting tissues and organs from the site of donation to the patient in need, while maintaining viability, is a limiting factor in transplantation medicine. One way in which the supply chain of organs for transplantation can be improved is to discover novel approaches and technologies that preserve the health of organs outside of the body. The dominant technologies that are currently in use in the supply chain for biological materials maintain tissue temperatures ranging from a controlled room temperature (+25 °C to +15 °C) to cryogenic (-120 °C to -196 °C) temperatures (reviewed in Criswell et al. Stem Cells Transl Med. 2022). However, there are many cells and tissues, as well as all major organs, that respond less robustly to preservation attempts, particularly when there is a need for transport over long distances that require more time. In this perspective article, we will highlight the current challenges and advances in biopreservation aimed at "freezing biological time," and discuss the future directions and requirements needed in the field.

Shipping and Logistics Considerations for Regenerative Medicine Therapies.

Advances in regenerative medicine manufacturing continue to be a priority for achieving the full commercial potential of important breakthrough therapies. Equally important will be the establishment of distribution chains that support the transport of live cells and engineered tissues and organs resulting from these advanced biomanufacturing processes. The importance of a well-managed distribution chain for products requiring specialized handling procedures was highlighted during the COVID-19 pandemic and serves as a reminder of the critical role of logistics and distribution in the success of breakthrough therapies. This perspective article will provide insight into current practices and future considerations for creating global distribution chains that facilitate the successful deployment of regenerative medicine therapies to the vast number of patients that would benefit from them worldwide.

Pituitary Lineage Differentiation from Human Induced Pluripotent Stem Cells in 2D and 3D Cultures.

Despite its small size, the pituitary gland plays a central role in the maintenance of normal homeostasis of most physiological systems through its regulation of the function of other endocrine glands. The complexity of the anterior pituitary gland, due to its composition of several different hormone-secreting cell types, begets a plethora of disorders and pathologies due primarily to hyposecretion or hypersecretion of hormones. The gonadotrophs, which make up less than 5% of the total number of cells in the anterior pituitary, serve to regulate gonad development and sexual reproduction in males and females. Despite the increased research on the development of models to study pituitary function within the last decade, a model specifically designed to study the gonadotrophs is still lacking. The development of organoid technology has facilitated research in the field of personalized medicine and physiological testing using patient-derived cells. The ability to produce pituitary organoids would allow researchers to construct an in vitro model of the human hypothalamic-pituitary-gonadal or hypothalamic-pituitary-adrenal axis to use in further fertility or endocrine research. The application of this technology in patients could revolutionize the treatment of infertility and a variety of neuroendocrine disorders. The impetus behind this study was to develop a pituitary-like organoid consisting only of gonadotrophs. Despite the lack of success in differentiating gonadotrophs, pituitary-like organoids were differentiated from human-induced pluripotent stem cells. In addition, two-dimensional and three-dimensional differentiated cultures were characterized and compared to human adult cadaveric pituitary tissue.

Development of a Rat Model of Fasciotomy Treatment for Compartment Syndrome.

Skeletal muscle injuries are a major cause of disability for military and civilian populations. Compartment syndrome (CS) in skeletal muscle results from an edema-induced increase in intracompartmental pressure (ICP) after primary injury. Untreated ICP will occlude the tissue vasculature, tissue necrosis, and potential loss of limb. The current standard of care for CS is surgical fasciotomy, an incision through the muscle fascia to relieve ICP. Early fasciotomy will preserve the limb, but often leaves patients with long-term scarring and reduced muscle function. Our group previously developed and characterized a rat model of CS to explore the pathophysiology of CS and test new therapies. We present an expansion of this CS model, including the fasciotomy, to better simulate clinical treatment. CS was induced on the hind limb of adult male Lewis rats and fasciotomy was performed 24 h later. Less than 20% of the rats that underwent fasciotomy showed detectable force 4 days after injury, compared with the 75% of rats that underwent CS induction without fasciotomy. Muscles undergoing fasciotomy showed a significant increase in fibrosis and an increased number of macrophages, Pax7+ satellite cells, and α-smooth muscle actin+ myofibroblasts at 7 days postinjury. These data indicate that the use of fasciotomy in a rat model of CS resulted in injury sequelae that reflect the severity of human clinical disease presentation along with current standard of care. Impact Statement Current animal models of skeletal muscle injury struggle to accurately reflect the injury sequelae seen in humans, particularly in rats and mice. These animals also recover faster than humans do. More accurate recapitulation of the injury is needed to better study the injury progression, as well as screen for novel therapies. This research combines an existing model of compartment syndrome with its clinical standard of care (fasciotomy), creating a more accurate rat model of injury, and providing for a better treatment screening tool. These results show how our model leads to a sustained skeletal muscle deficit with increased inflammation.

Bioreactor design and validation for manufacturing strategies in tissue engineering.

The fields of regenerative medicine and tissue engineering offer new therapeutic options to restore, maintain or improve tissue function following disease or injury. To maximize the biological function of a tissue-engineered clinical product, specific conditions must be maintained within a bioreactor to allow the maturation of the product in preparation for implantation. Specifically, the bioreactor should be designed to mimic the mechanical, electrochemical and biochemical environment that the product will be exposed to in vivo. Real-time monitoring of the functional capacity of tissue-engineered products during manufacturing is a critical component of the quality management process. The present review provides a brief overview of bioreactor engineering considerations. In addition, strategies for bioreactor automation, in-line product monitoring and quality assurance are discussed.

Sustained therapeutic effect of an anti-inflammatory peptide encapsulated in nanoparticles on ocular vascular leakage in diabetic retinopathy.

Pigment epithelium-derived factor (PEDF), an endogenous Wnt signaling inhibitor in the serine proteinase inhibitors (SERPIN) super family, is present in multiple organs, including the vitreous. Significantly low levels of PEDF in the vitreous are found to associate with pathological retinal vascular leakage and inflammation in diabetic retinopathy (DR). Intravitreal delivery of PEDF represents a promising therapeutic approach for DR. However, PEDF has a short half-life after intravitreal injection, which represents a major hurdle for the long-term treatment. Here we report the prolonged therapeutic effects of a 34-mer peptide of the PEDF N-terminus, encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (PEDF34-NP), on DR. PEDF34-NP inhibited hypoxia-induced expression of vascular endothelial growth factor and reduced levels of intercellular adhesion molecule 1 (ICAM-1) in cultured retinal cells. In addition, PEDF34-NP significantly ameliorated ischemia-induced retinal neovascularization in the oxygen-induced retinopathy rat model, and significantly reduced retinal vascular leakage and inflammation in streptozotocin-induced diabetic rats up to 4 weeks after intravitreal injection, as compared to PLGA-NP control. Intravitreal injection of PEDF34-NP did not display any detectable toxicities to retinal structure and function. Our findings suggest that PEDF34-NP can confer sustained therapeutic effects on retinal inflammation and vascular leakage, having considerable potential to provide long-term treatment options for DR.

Enhanced method to select human oogonial stem cells for fertility research.

Alternative methods to obtain mature oocytes are still needed for women with premature ovarian failure (POF). Oogonial stem cells (OSCs), found in adult ovaries, have provided insight into potential paths to treating infertility. Previously, the DDX4 antibody marker alone was utilized to isolate OSCs; however, extensive debate over its location in OSCs versus resulting oocytes (transmembrane or intracytoplasmic) has raised doubt about the identity of these cells. Separate groups, however, have efficiently isolated OSCs using another antibody marker Ifitm3 which is consistently recognized to be transmembrane in location. We hypothesized that by using anti-DDX4 and anti-IFITM3 antibodies, in combination, with MACS, we would improve the yield of isolated OSCs versus using anti-DDX4 antibodies alone. Our study supports earlier findings of OSCs in ovaries during the entire female lifespan: from reproductive age through post-menopausal age. MACS sorting ovarian cells using a the two-marker combination yielded a ~ twofold higher percentage of OSCs from a given mass of ovarian tissue compared to existing single marker methods while minimizing the debate surrounding germline marker selection. During in vitro culture, isolated cells retained the germline phenotype expression of DDX4 and IFITM3 as confirmed by gene expression analysis, demonstrated characteristic germline stem cell self-assembly into embryoid bodies, and formed > 40 µm "oocyte-like" structures that expressed the early oocyte markers GDF9, DAZL, and ZP1. This enhanced and novel method is clinically significant as it could be utilized in the future to more efficiently produce mature, secondary oocytes, for use with IVF/ICSI to treat POF patients.

Regenerative Medicine Approaches in Bioengineering Female Reproductive Tissues.

Diseases, disorders, and dysfunctions of the female reproductive tract tissues can result in either infertility and/or hormonal imbalance. Current treatment options are limited and often do not result in tissue function restoration, requiring alternative therapeutic approaches. Regenerative medicine offers potential new therapies through the bioengineering of female reproductive tissues. This review focuses on some of the current technologies that could address the restoration of functional female reproductive tissues, including the use of stem cells, biomaterial scaffolds, bio-printing, and bio-fabrication of tissues or organoids. The use of these approaches could also be used to address issues in infertility. Strategies such as cell-based hormone replacement therapy could provide a more natural means of restoring normal ovarian physiology. Engineering of reproductive tissues and organs could serve as a powerful tool for correcting developmental anomalies. Organ-on-a-chip technologies could be used to perform drug screening for personalized medicine approaches and scientific investigations of the complex physiological interactions between the female reproductive tissues and other organ systems. While some of these technologies have already been developed, others have not been translated for clinical application. The continuous evolution of biomaterials and techniques, advances in bioprinting, along with emerging ideas for new approaches, shows a promising future for treating female reproductive tract-related disorders and dysfunctions.

Engineering Functional Rat Ovarian Spheroids Using Granulosa and Theca Cells.

Although menopausal hormone therapy (MHT) is the most effective approach to managing the loss of ovarian activity, serious side effects have been reported. Cell-based therapy is a promising alternative for MHT. This study constructed engineered ovarian cell spheroids and investigated their endocrine function. Theca and granulosa cells were isolated from ovaries of 10-week-old rats. Two types of engineered ovarian cell spheroids were fabricated through forced aggregation in microwells, multilayered spheroids with centralized granulosa aggregates surrounded by an outer layer of theca cells and mixed ovarian spheroids lacking spatial rearrangement. The ovarian cell spheroids were encapsulated into a collagen gel. Non-aggregated ovarian cells served as controls. The endocrine function of the engineered ovarian spheroids was assessed over 30 days. The structure of the spheroids was well maintained during culture. The secretion of 17ß-estradiol from both types of engineered ovarian cell spheroids was higher than in the control group and increased continuously in a time-dependent manner. Secretion of 17ß-estradiol in the multi-layered ovarian cell spheroids was higher than in the non-layered constructs. Increased secretion of progesterone was detected in the multi-layered ovarian cell spheroids at day 5 of culture and was sustained during the culture period. The initial secretion level of progesterone in the non-layered ovarian cell spheroids was similar to those from the controls and increased significantly from days 21 to 30. An in vitro rat model of engineered ovarian cell spheroids was developed that was capable of secreting sex steroid hormones, indicating that the hormone secreting function of ovaries can be recapitulated ex vivo and potentially adapted for MHT.

3-D Human Renal Tubular Organoids Generated from Urine-Derived Stem Cells for Nephrotoxicity Screening.

The development of human cell-based systems to replace the use of rodents or the two-dimensional culture of cells for studying nephrotoxicity is urgently needed. Human urine-derived stem cells were differentiated into renal tubular epithelial cells in three-dimensional (3-D) culture after being induced by a kidney extracellular matrix. Levels of CYP2E1 and KIM-1 in 3-D organoids were significantly increased in response to acetone and cisplatin. This 3-D culture system provides an alternative tool for nephrotoxicity screening and research.

Microfluidic Systems for Assisted Reproductive Technologies: Advantages and Potential Applications.

Microfluidic technologies have emerged as a powerful tool that can closely replicate the in-vivo physiological conditions of organ systems. Assisted reproductive technology (ART), while being able to achieve successful outcomes, still faces challenges related to technical error, efficiency, cost, and monitoring/assessment. In this review, we provide a brief overview of the uses of microfluidic devices in the culture, maintenance and study of ovarian follicle development for experimental and therapeutic applications. We discuss existing microfluidic platforms for oocyte and sperm selection and maintenance, facilitation of fertilization by in-vitro fertilization/intracytoplastimc sperm injection, and monitoring, selection and maintenance of resulting embryos. Furthermore, we discuss the possibility of future integration of these technologies onto a single platform and the limitations facing the development of these systems. In spite of these challenges, we envision that microfluidic systems will likely evolve and inevitably revolutionize both fundamental, reproductive physiology/toxicology research as well as clinically applicable ART.

N-Acetyl-L-Cysteine Reduces Fibrosis and Improves Muscle Function After Acute Compartment Syndrome Injury.

INTRODUCTION: Upon injury, skeletal muscle undergoes a multiphase process beginning with degeneration of the damaged tissue, which is accompanied by inflammation and finally regeneration. One consequence of an injured microenvironment is excessive production of reactive oxygen species, which results in attenuated regeneration and recovery of function ultimately leading to fibrosis and disability. The objective of this research was to test the potential of the antioxidant, N-Acetyl-L-Cysteine (NAC), as a mediator of reactive oxygen species damage that results from traumatic muscle injury in order to support repair and regeneration of wounded muscle tissue and improve function recovery. MATERIALS AND METHODS: Adult female Lewis rats were subjected to compartment syndrome injury as previously published by our group. Rats received intramuscular injections of NAC or vehicle at 24, 48, and 72 hours postinjury. Muscle function, tissue fibrosis, and the expression of myogenic and angiogenic markers were measured. RESULTS: Muscle function was significantly improved, and tissue fibrosis was significantly decreased in NAC-treated muscles. CONCLUSIONS: These results suggest that NAC treatment of skeletal muscle after injury may be a viable option for the prevention of long-term fibrosis and scar formation, facilitating recovery of muscle function.

A cocktail of growth factors released from a heparin hyaluronic-acid hydrogel promotes the myogenic potential of human urine-derived stem cells in vivo.

Traditional cell therapy technology relies on the maximum expansion of primary stem cells in vitro, through multiple passages and potential differentiation protocols, in order to generate the abundance of cells needed prior to transplantation in vivo. Implantation of in vitro over-expanded and pre-differentiated cells typically results in poor cell survival and reduced regeneration capacity for tissue repair in vivo. We hypothesized that implantation of primary stem cells, after a short time culture in vitro (passage number ≤p3), in combination with controlled release of relevant growth factors would improve in vivo cell viability, engraftment and tissue regeneration. The goal of this study was to determine whether the release of myogenic growth factors from a heparin-hyaluronic acid gel (hp-HA gel) could enhance in vivo cell survival, in-growth and myogenic differentiation of human urine-derived stem cells (USC) with a corresponding enhancement in graft vascularization, innervation and regenerative properties. Human USC were obtained from healthy adult donors (n = 6), expanded and then mixed with a hp-HA gel containing sets of growth factors known to enhance myogenesis (IGF1, HGF, PDGF-BB), neurogenesis (NGF, FGF) and angiogenesis (VEGF), or a cocktail with a combination of growth factors. Primary cultured USC (p3) mixed with the hp-HA gel and the various combinations of growth factors, were subcutaneously injected into athymic mice. In vivo cell survival, engraftment and functional differentiation within the host tissue were assessed. The implanted grafts containing USC and the growth factor cocktail showed the greatest number of surviving cells as well as increased numbers of cells that expressed myogenic and endothelial cell markers as compared to other groups 4 weeks after implantation. Moreover, the graft with USC and the growth factor cocktail showed increased numbers of blood vessels and infiltrating neurons. Thus, growth factors released in a controlled manner from an hp-HA gel containing USC efficiently improved in vivo cell survival and supported vascularization and myogenic differentiation within the grafts. This study provides evidence for the use of primary USC and growth factors in a hydrogel as a novel mode of cell therapy for the promotion of myogenic differentiation for the treatment of injured muscle tissue. STATEMENT OF SIGNIFICANCE: Cell therapies are a promising treatment option for neuromuscular dysfunction disorders. However, major limitations in cell retention and engraftment after implantation remain a hindrance to the use of stem cell therapy for the treatment of muscle injuries or diseased tissues. Implanted long-term in vitro cultured cells tend to demonstrate low rates of survival and tissue engraftment, lessened paracrine effects, and poor homing and differentiation. Human USC are an easily obtainable stem cell source that possess stem cell characteristics such as a robust proliferative potential, paracrine effects on neighboring cells, and multi-potential differentiation. In this study, we demonstrated that a combination of primary human USC with a cocktail of growth factors combined in a hyaluronic gel was optimal for cell survival and engraftment, including myogenic differentiation potential of USC, angiogenesis and host nerve fiber recruitment in vivo. The present study also demonstrated that the use of primary urine derived stem cells at early passages, without in vitro pre-differentiation, implanted in a hyaluronic-heparin hydrogel containing a cocktail of growth factors, provided an alternative safe site-specific delivery method for cell therapy.

The Use of Pulsed Electromagnetic Field to Modulate Inflammation and Improve Tissue Regeneration: A Review.

Pulsed electromagnetic field (PEMF) is emerging as innovative treatment for regulation of inflammation, which could have significant effects on tissue regeneration. PEMF modulates inflammatory processes through the regulation of pro- and anti-inflammatory cytokine secretion during different stages of inflammatory response. Consistent outcomes in studies involving animal and human tissue have shown promise for the use of PEMF as an alternative or complementary treatment to pharmaceutical therapies. Thus, PEMF treatment could provide a novel nonpharmaceutical means of modulating inflammation in injured tissues resulting in enhanced functional recovery. This review examines the effect of PEMF on immunomodulatory cells (e.g., mesenchymal stem/stromal cells [MSCs] and macrophages [MΦ]) to better understand the potential for PEMF therapy to modulate inflammatory signaling pathways and improve tissue regeneration. This review cites published data that support the use of PEMF to improve tissue regeneration. Our studies included herein confirm anti-inflammatory effects of PEMF on MSCs and MΦ.

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling.

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.

* The Impact of Age on Skeletal Muscle Progenitor Cell Survival and Fate After Injury.

Sarcopenia is defined as the loss of skeletal muscle mass and function due to age, and represents a major cause of disability in the elderly population. The contributing factors to the onset of sarcopenia are not well defined, but appear to involve age-dependent changes in both the tissue microenvironment and muscle progenitor cell (MPC) population. MPC transplantation has the potential to be a novel therapy for treatment of muscle dysfunction due to aging or injury, but has not shown significant clinical efficacy to date. The goal of this research was to use a rat model of skeletal muscle injury to examine the differential effects of age on MPC survival, differentiation, and tissue regeneration after transplantation. Fluorescently labeled MPCs, derived from young (YMPCs) and adult (AMPCs) donor rats, were transplanted in the injured tibialis anterior (TA) muscles of young, adult, and aged rats. Our results demonstrated that integration and maturation of YMPCs into mature myofibers were dependent on the age of the host microenvironment; whereas, the integration and maturation of AMPCs were less dependent on age and more dependent on intrinsic cellular changes. These data suggest that the age of both the host microenvironment and cells for transplantation must be considered when designing cell therapy regimens.